Calcium pyrophosphate dihydrate (CPPD) deposition disease is an arthropathy characterized by the accumulation of CPPD crystals in articular tissues including synovial fluid. CPPD crystals contribute significantly to the chronic pain and tissue damage of joint degeneration, and in vitro they induce neutrophil activation and fibroblast and chondrocyte mitogenesis as well as the production of matrix metalloproteinases (MMP) and prostaglandins. CPPD deposition is associated with acute inflammatory episodes (pseudogout), chronic arthritis, and degenerative joint disease. Although only about 10% of the CPPD patient population ever experience acute inflammatory attacks, the majority of patients with chronic arthritis of the large joints have CPPD deposition. CPPD crystals play a significant role in arthritic disease progression. Synovial fluids containing CPPD crystals sampled from patients with degenerative joint disease have high concentrations of cartilage fragments and MMPs, e.g., collagenase and stromelysin. (Swan, A. B. et al. (1994) Ann. Rheum. Dis. 53:467-470; Lohmander, L. S. et al. (1993) Arthritis. Rheum. 36:181-189.)
Deposition of CPPD crystals appears to be related to excess levels of extracellular calcium, pyrophosphate (PPi), or both. Whereas elevated calcium levels do not appear to be a major contributing factor to CPPD deposition in joints, elevated PPi levels have been observed in the synovial fluids from patients with CPPD deposition. Synovial fluid PPi may be produced by joint tissues because PPi levels are higher in synovial fluid than in plasma and in vitro cartilage explants release PPi into the extracellular medium. (Ryan, L. M. et al. (1996) J. Rheumatol. 23:214-219.)
Enzymes that hydrolyze nucleotide triphosphates and release PPi are called nucleotide pyrophosphohydrolases (NTPPH). NTPPH activity is found in synovial fluid and correlates with the production of PPi. Elevated ATP levels have been found in joint fluids of patients with CPPD deposition, and addition of extracellular ATP to joint tissues and fluids results in the production of PPi. (Park, W. I. et al. (1996) J. Rheumatol. 23:665-671.) The levels of molecules with NTPPH activity are higher in extracts from cartilage containing CPPD crystals than from cartilage lacking crystals. Matrix vesicles released from articular cartilage in vitro show high NTPPH activity and produce CPPD in the presence of calcium and ATP. (Derfus, B. A. et al. (1992) Arthritis. Rheum. 35:231-240.)
A protein demonstrating NTPPH activity and having a molecular weight of 61 kD was recently purified from porcine articular cartilage explant conditioned medium. (Masuda, I. et al. (1995) J. Clin. Invest. 95:699-704.) The first 26 residues of the amino-terminus were sequenced and showed no homology to any protein in public databases. Antipeptide antibodies were generated against the 61 kD porcine protein, and the antisera identified the original 61 kD protein and an additional 127 kD vesicle-associated protein in conditioned medium from cultures of both chondrocytes and cartilage explants. The 61 kD isoform is believed to be a catalytically active proteolytic fragment of the 127 kD protein. Both the 61 kD and the 127 kD isoforms were identified in human synovial fluids, and a 100 kD protein was identified in human serum. Using the antipeptide antibody on immunoblots of tissue extracts, NTPPH expression was found only in articular tissues, e.g., hyaline cartilage, fibrocartilage, tendon, and ligament, in which CPPD deposition occurs. (Cardenal, A. et al. (1996) Arthritis Rheum. 39:252-256; Cardenal, A. et al. (1996) Arthritis Rheum. 39:245-251.) Recently, a partial porcine NTPPH cDNA was isolated. (Masuda, I. et al. (1997) Gene 197:227-282.)
A full length human nucleotide pyrophosphohydrolase (NTPPH-1) has been cloned using a cDNA clone isolated from a cartilage cDNA library. Northern analysis of human, dog, and rabbit joint tissue RNA samples indicated elevated levels of NTPPH-1 expression in articular cartilage, and lower, but significant, levels of expression in synovium, meniscus, tendon and ligament. Expression studies on additional human tissues demonstrated significant mRNA levels in skeletal muscle, heart muscle, and bone marrow; and lower, but detectable, levels in trachea, spinal cord, thyroid, stomach, testis, uterus, small intestine, colon, thymus, placenta, lymph, and adrenal tissue.
The discovery of a new human nucleotide pyrophosphohydrolase, NTPPH-2, and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of arthropathies, immunological disorders, and cancers.